JPS62134083A - Iron-containing microorganism and production thereof - Google Patents

Abstract

PURPOSE:To accumulate iron in a high concentration and produce microbial cells to be very useful iron agent in supplying iron, by cultivating a nontoxic microorganism in a culture medium containing an added iron compound. CONSTITUTION:An iron compound, e.g. iron butyrate, ferrous chloride or ferric sulfate, etc., in an amount of preferably >=0.5wt% based on a carbon source in a culture medium expressed in terms of metallic iron is added to the culture medium containing at least the carbon source and a nitrogen source to adjust the magnesium concentration in the culture medium to <=10mg/l expressed in terms of metallic magnesium. A nontoxic microorganism, e.g. yeast, lactic acid bacterium, Bacillus subtilis, etc., is inoculated into the above-mentioned culture medium and cultivated to collect the aimed microbial cells containing >=100mg based on 100g dried microbial cells expressed in terms of metallic iron.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to microbial cells 1 which accumulate iron at high concentrations and become extremely useful iron supplements when used for iron supplementation.

It is said that due to changes in dietary habits, the number of people with 11 bloodstream blood clots is on the rise, and to counter this problem, it is desirable to supplement iron from natural sources, that is, from daily meals. Even in animal or plant foods, which are said to have a large amount of iron, the iron content is as low as 0.1 IOB per 100 g of edible portion, so supplementation with iron supplements is inevitable. Conventional iron preparations are often made into sustained-release formulations for the purpose of alleviating gastrointestinal disorders and improving absorption, and they are formulated with organic polymer compounds (e.g. dextran, carboxymethyl cellulose,
Mudai iron salts are dispersed in methyl acrylate/methacrylic acid copolymers, carboxyvinyl polymers, etc.).

However, high-molecular compounds are indigestible, and iron salts are not well absorbed, requiring large amounts to be administered, for example, as metal iron. /day), this often results in vomiting and diarrhea, and in the case of elderly people, there are undesirable cases in which indigestible organic polymer compounds remain in the stomach for a long time.

The inventors of the present invention have conducted studies to solve the problems mentioned in 1-1, conducted research to increase the concentration of iron in microbial cells, added iron compounds at high concentrations, and The present invention was completed based on the finding that a significantly large amount of iron accumulates in microorganisms by culturing whole organisms in a medium containing limited amounts of iron.

Does this invention increase iron content? The first objective is to provide microbial cells that have a high agricultural yield, and the second objective is to provide an industrially advantageous method for producing such microbial cells. According to the first aspect of the present invention, the purpose of L t51 is to use iron as a metal, which is obtained by culturing microorganisms with the addition of an iron compound in a medium containing at least a carbon source and a nitrogen source. The second object is achieved by microbial cells containing at least 100+ ng of iron per 100 Fi of dry cells, according to the second aspect of the present invention, containing at least a carbon source and a nitrogen source, and containing an iron compound. This is achieved by culturing the microorganism in the added medium with the concentration of magnesium in the medium below J O + ng/l to produce a microorganism containing a high concentration of iron.

The present invention will be explained in detail below.

The microorganisms used in the present invention are microorganisms that are harmless even if eaten, and suitable examples include yeasts such as those of the genus Satucharomyces, lactic acid bacteria, Bacillus subtilis, and the like, with yeast being particularly preferred.

The carbon source used in the culture medium of the present invention may be one that can be assimilated by microorganisms, such as saccharides, isoic acid, fats and oils, and soybean acid.
It can be anything. Nitrogen sources include meat extract, peptone, yeast extract, other proteins, their hydrolysates, organic nitrogen-containing compounds such as amino acids, Kuroiso nitrogen-containing compounds such as ammonia, ammonium salts, nitrates, etc.
Any nitrogen source conventionally used for culturing whole organisms can be used.

In order to produce the microorganism of the present invention containing iron at a high concentration of 2, the microorganism medium needs to have a magnesium content of 1 O+I1 g/l or less in terms of metallic magnesium. Immediately, the amount of magnesium in the medium is 10 mg.
/l or less, the iron content accumulated in the producing microorganisms increases significantly.

Conventionally, when culturing microorganisms, a magnesium compound such as magnesium sulfate has been added to encourage their growth. For example, magnesium is contained in the medium as metal magnesium at 0.1 to 0.2 g/l. In the present invention, as described above, the amount of magnesium in the medium is limited, but since there are substances containing magnesium in the medium components, such as polypeptone and yeast extract, the magnesium content in the medium components must be increased in advance. Flowers are measured and if the amount of magnesium is present in excess of the limit value, h! It is necessary to reselect the types and amounts of the components to bring them within the limits of the present invention. On the other hand, when the magnesium content derived from the medium components is small within the limit value, a magnesium compound such as magnesium sulfate may be added within the range not exceeding the limit value.

Next, in order to obtain the microorganism of the present invention, it is desirable to add iron to the medium from the early stage of culture.

The iron compound to be added may be organic iron such as iron butyrate or iron heptomalate, or non-ferrous iron such as ferrous chloride, ferrous ammonium sulfate, or ferrous bromide, but the latter is preferable. is economical. The amount of iron compound added in the medium is preferably 0.5% by weight or more of the weight of the carbon source used in the medium, calculated as metallic iron.

Fermentation proceeds under aerobic conditions, such as in a shaking medium g1. Culture conditions such as culture temperature and medium pH should be the conditions normally used for the microorganism used (usually the culture temperature is 15-=-35°C, l). The culture time is about 24 to 48 hours, depending on the composition of the medium and the culture method, and usually 24 hours is sufficient.

M of iron per weight of microorganism cells obtained in this way
The accumulation tends to increase as the magnesium content in the medium decreases, but as the magnesium content decreases,
Since bacterial cell production +i1.6 decreases, in commercial implementation, the selection should be made based on both factors.After culturing as described above, collect the bacterial cells by the usual method, The product is washed with water or an aqueous solution of nonodium salt of ethylenenoamine titraacetic acid (hereinafter abbreviated as EDTA salt), and compressed or dried to obtain a product.

To determine the amount of iron contained in bacterial cells, soak the bacterial cells in water and then add 10+++
El') TA salt of M was washed with aqueous solution of TA salt, and this was carefully incinerated under intense heat. After extraction with f3N-HCI, the Matsubara method using ortho-7 enanthroline as a coloring agent [Journal of the Japanese Society of Hematology] , Dan, 434 (1962)].

The iron-containing microorganism cells of the present invention obtained by the above culture are accumulated in about 30 times as much water as in the wet mold, and in the same amount as organic iron compounds that are difficult to separate from the cells. It is speculated that this may be the case. The amount of iron contained in the microorganisms of the present invention (in terms of metallic iron) is approximately 201 iron per 100g in conventional dry baker's yeast, but in Sushi, +iλ 1 per 100) r of dried bacterial cells
00~750m1? is significantly high.

(Examples) Next, the present invention will be described in detail, but the present invention is not limited to these examples.

Example 1 Glucose 20g/l, polypeptone 20g/l, yeast extract 10l? A medium containing G+tg/l (derived from polypeptone and magnesium contained in yeast extract, and containing G+tg/l as metallic magnesium).
(contains magnesium content of I) 50ml
After heating and sterilizing, inoculate baker's yeast [Oriental Acid @ manufactured by Co., Ltd. 1] and inoculate at 30℃ for 2 hours.
Culture was performed for 4 hours. In this way, a preculture solution is prepared. Add ferrous ammonium sulfate to 0.1% by weight as a medium with a unidirectional composition (the amount of ferrous ammonium sulfate added is 0.7% by weight as metallic iron when considered as a carbon source for the medium). A 500 [Ql volume Erlenmeyer flask containing 55 ml of the prepared medium was inoculated with the above preculture solution at a ratio of 2%, and main culture was carried out at 30°C with shaking culture at 24 [1:'l-rll'l]. I did it. After main culture, culture 1! The liquid was centrifuged at 4°C to separate the bacterial cells, which were washed three times with 20 ml of ion-exchanged water, and then added with a 10+oM EDTA salt aqueous solution 2
The iron-containing microorganism cells of the present invention were obtained by washing three times with 0+nl.

The obtained bacterial cells weighed 0.62 g as a dry cell.

As a result of measuring the iron concentration of this material using the Matsubara method described above,
The iron content was 334 tons per 100 g of dry bacterial cells.

Example 2 The medium for main culture was 20 g/I of glucose and 5 I of ammonium sulfate. /l, potassium dihydrogen phosphate 0.85g/l
, dipotassium hydrogen phosphate 0.15 g/l, sodium chloride 0.18/l, calcium chloride (dihydrate) 0.1 g
/I, biotin 20 μf/1, calcium pantothenate 2 tng/l 1 folic acid 2 μg/l, inositol 1 (IB/l, niacin 400 μs/I, p-
jilX400μFl/L') Bo7 Rabin 200ug
/I, thiamine hydrochloride 400μFI/I, yeast extract 5g/! (This medium is derived from the magnesium contained in the yeast extract and contains 1,511,111?/l of magnesium as metallic magnesium.) The iron content of the iron-containing microorganism cells was 772 per 100 g of bread yeast @ dried cells using the same procedure as in Example 1.
It was B.

Example 3 The medium for main culture was glucose 2o/l, polypeptone 108/1, yeast extract 5g/l, Ell Pi
A medium containing 1 g/l of ferrous ammonium (this is derived from the magnesium contained in polypeptone and yeast extract, and contains 3 mg/l as metallic magnesium).
The culture was carried out in the same manner as in Example 1, except that the method was changed to (containing magnesium). The electric charge of the dry 1W body obtained in this way is (1,745R body 100I?/457
It was 111R.

Example 4 Magnesium sulfate was newly added to the main culture medium used in Example 3 to bring the final concentration of magnesium to 8 InH/
(The medium used in Example 3 is polished with magnesium contained in polypeptone and yeast extract, and the medium contains 3/1 magnesium, so this magnesium is not included. , refers to the magnesium content in the medium. Also in the comparative examples described later, it has the same meaning as the final concentration as magnesium). Other than that, culturing was performed in the same manner as in Example 3. The amount of dried bacterial cells thus obtained was 0.58 g, and the iron content was 169+ng per 100 g of baker's yeast dried bacterial cells.

Comparative Example 1 The same operation as in Example 3 was performed except that magnesium sulfate was added to the main culture medium used in Example 3 to make the final concentration of magnesium 15B/1. The amount of dried bacterial cells obtained was 60 g, and the iron content was 88 B/100 g of dried bacterial cells.

Comparative Example 2 The same operation as in Example 3 was performed, except that magnesium sulfate was added to the main culture medium used in Example 3 to give a final concentration of magnesium of 30 mg/11. The amount of dried bacterial cells obtained was 0.62 g, and the iron content was 71 mg per OOg of dried bacterial cells.

Add aluminum to make the final concentration as magnesium 50+ng/l.
The same operation as in Example 3 was performed, except that a culture medium having been prepared by The resulting dry bacterial mass was 0.59g.
C'', its iron content is 89+ per 100g of dry bacterial cells.
It was IIg.

Comparative Example 4 Magnesium sulfate was added to the main culture medium used in Example 3, and the final concentration of magnesium was 10.

m+? The same repeated operations as in Example 3 were carried out, except that a medium adjusted to 1/l was used. As a result, the amount of dried bacterial cells obtained was 61 g of O, and the iron content was 69+ng per 100 g of dry bacterial cells.

Comparative Example 5 The same cultivation as in Example 3 was carried out, except that magnesium sulfate was added to the main culture medium used in Example 3 to give a final magnesium concentration of 200 mg/l. The amount of dried bacterial cells obtained was 0.60 g, and the iron content was 81B per 100 g of dry bacterial cells. Example 5 The amount of dried bacterial cells was 0.72 g, and the iron content was The amount of drunkenness was 3621D per 100B of dry bacterial cells.

Example 6 A medium consisting of 5 g/l of hefton, 3 g/l of yeast extract, and 3 g/l of malt extract (this medium contains polypeptone and the magnesium content contained in yeast extract Polished rice contains 1.7111g/l of magnesium)
All operations were performed in the same manner as in Example 1, except that the lamp was turned on instead of . As a result, the amount of dried bacterial cells obtained was 0.22g.
The iron content is 100g of dried beer yeast cells.
It was 43,310g per serving.

Example 7 As a microorganism, Bacillus subtilis IAM1076
The preculture medium was 10 g/l of tryptone, 5 g/l of yeast extract, 10 g/l of salt, and 18 g/l of glucose.
All procedures were carried out as in Example 1 except that the medium was changed to a medium consisting of /1.
A similar repetition was performed. As a result, the iron content of the obtained bacterial cells was 360+ng per 100 g of dry bacterial cells.

(Effect of the invention) Since the conventional sustained-release iron preparation is made of indigestible Aiso polymer compound, it uses digestible natural bacterial cells as a carrier, so iron is gradually released as it is digested. , side effects such as gastrointestinal disorders can be prevented. It is also assumed that iron exists in the form of organic iron, which is better absorbed than the conventional iron rule. In addition, when yeast, bacteria, lactic acid bacteria, etc. are used as microorganisms, the bacterial cells themselves contain various vitamins, minerals, proteins, sugars, etc., and together with slow release, iron absorption is further improved. Therefore, the microbial cells of the present invention containing iron at high concentrations are considered to be extremely effective for people with anemia.

Claims (2)

[Claims] (1) Obtained by culturing microorganisms with the addition of an iron compound in a medium containing at least a carbon source and a nitrogen source.
Microbial cells containing mg or more. (2) Cultivate microorganisms in a medium containing at least a carbon source and a nitrogen source and supplemented with an iron compound, with the concentration of magnesium in the medium being 10 mg/l or less as metallic magnesium, and containing a high concentration of iron. A method for producing microorganisms that